mRNAs that mature through trans-splicing in Caenorhabditis elegans have a trimethylguanosine cap at their 5' termini.

Abstract

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.Eucaryotic mRNAs possess modified 5' termini of the form m7G(5')ppp(5')N (monomethylguanosine). This modification, known as a cap, is important in binding factors involved in the initiation of translation (for reviews, see references 19 and 20). The cap influences splicing of premRNA and contributes to the stability of mRNA (4,5,10,11,16,17).Small nuclear RNAs (snRNAs) are an important component of the splicing machinery in eucaryotes (for reviews, see references 7 and 21). These RNAs are complexed with proteins into small nuclear ribonucleoprotein particles (for reviews, see references 14 and 15). snRNAs have a trimethylguanosine cap [m32 2.7G(5')ppp(5')N] at their 5' ends (for a review, see reference 18) and are not translated.Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through an unusual mechanism in which the 5' 22 nucleotides of the mRNA are acquired through a trans-splicing reaction (1,12). This reaction involves a unique small nuclear ribonucleoprotein particle containing spliced leader (SL) RNA (2,22,23). SL RNA provides the 5' exon (the SL) in the trans-splicing reaction and is thought to act as its own Ul snRNA during the trans-splicing reaction (2). SL RNA also possesses the trimethylguanosine cap that is characteristic of eucaryotic snRNAs (22,23).Because of the importance of caps in mRNA maturation and translation, we examined whether the trimethylguanosine cap on the SL RNA was transferred to and retained on trans-spliced mRNAs in C. elegans. RNA purified from a mixed population of C. elegans containing all developmental stages was immunoprecipitated by using the monoclonal antibody recognizing trimethylguanosine (as described by Van Doren and Hirsh [23] (Fig. 1A, lanes 6 and 7). Protection of the antisense SL RNA probe yielded a fragment of approximately 90 bases. The actin 1 gene is one of four C. elegans actin genes. Actin 1 mRNA acquired the SL through trans-splicing and was immunoprecipitated by the monoclonal antibody, showing that it has a trimethylguanosine cap (Fig. 1B, lanes 6 and 7). Actin 1 mRNA will protect 220 bases of the antisense actin 1 RNA probe. The actin 4 gene is the only member of the actin gene family that does not undergo the trans-splicing reaction. The fragment of the antisense RNA probe protected by actin 4 mRNA was 83 bases. In contrast to actin 1 mRNA, actin 4...

Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency

“…This modification is of particular interest because the mRNAs of some lower metazoans contain mRNAs of two types, those containing m 7 GTP and those containing m 3 2,2,7 GTP. mRNAs from ∼70% of the genes in Caenhorabditis elegans contain the m 3 2,2,7 Gp 3 G cap due to trans-splicing (Liou and Blumenthal 1990;van Doren and Hirsh 1990;Zorio et al 1994). In the present study, we found that m 3 2,2,7…”

Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs

“…Our findings suggest that PIMT expression increases the amount of TMG-containing unspliced/partially spliced HIV-1 RNAs that are then exported through the CRM-1 pathway and expressed into the corresponding proteins in the cytoplasm. Compatible with our results, in Caenorhabditis elegans and Ascaris lumbricoides, nematode mRNAs can acquire TMG caps by transsplicing, and in these settings, virtually all actin and ribosomal protein mRNAs are TMG-capped and loaded onto polysomes and are functionally translated with efficiency (59)(60)(61). Similarly, during the replication of togaviruses (e.g., Semliki Forest virus and Sindbis virus), late viral mRNAs acquire hypermethylated guanosine caps, and the expression of late proteins is proficient (21,22).…”

Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

“…Although not closely related, these nematodes both add the canonical SL1 spliced leader to the majority of their mRNAs (Maroney et al 1995). Biochemical and molecular genetic investigations have built up a detailed picture of the mechanism and biological significance of spliced leader addition to mRNA in nematodes (Bruzik et al 1988;Thomas et al 1988;Maroney et al 1990a,b;Van Doren and Hirsh 1990;Denker et al 1996Denker et al , 2002Ferguson et al 1996;Ferguson and Rothman 1999;Lall et al 2004;Cheng et al 2007;MacMorris et al 2007). The conservation of the SL1 primary sequence between C. elegans and A. suum suggests a functional constraint.…”